Fatty Acid Composition and Thermotropic Behavior of Glycolipids and Other Membrane Lipids of Ulva lactuca (Chlorophyta) Inhabiting Different Climatic Zones.

Increasing global temperatures are expected to increase the risk of extinction of various species due to acceleration in the pace of shifting climate zones. Nevertheless, there is no information on the physicochemical properties of membrane lipids that enable the adaptation of the algae to different climatic zones. The present work aimed to compare fatty acid composition and thermal transitions of membrane lipids from green macroalgae Ulva lactuca harvested in the Sea of Japan and the Adriatic Sea in summer. U. lactuca inhabiting the Adriatic Sea had bleached parts of thalli which were completely devoid of chloroplast glycolipids. The adaptation to a warmer climatic zone was also accompanied by a significant decrease in the ratio between unsaturated and saturated fatty acids (UFA/SFA) of membrane lipids, especially in bleached thalli. Hence, bleaching of algae is probably associated with the significant decrease of the UFA/SFA ratio in glycolipids. The decreasing ratio of n-3/n-6 polyunsaturated fatty acids (PUFAs) was observed in extra-plastidial lipids and only in the major glycolipid, non-lamellar monogalactosyldiacylglycerol. The opposite thermotropic behavior of non-lamellar and lamellar glycolipids can contribute to maintenance of the highly dynamic structure of thylakoid membranes of algae in response to the increasing temperatures of climatic zones.

Immunogenicity and Protective Activity of a Chimeric Protein Based on the Domain III of the Tick-Borne Encephalitis Virus E Protein and the OmpF Porin of Yersinia pseudotuberculosis Incorporated into the TI-Complex.

Tick-borne encephalitis (TBE) is a widespread, dangerous infection. Unfortunately, all attempts to create safe anti-TBE subunit vaccines are still unsuccessful due to their low immunogenicity. The goal of the present work was to investigate the immunogenicity of a recombinant chimeric protein created by the fusion of the EIII protein, comprising domain III and a stem region of the tick-borne encephalitis virus (TBEV) E protein, and the OmpF porin of Yersinia pseudotuberculosis (OmpF-EIII). Adjuvanted antigen delivery systems, the tubular immunostimulating complexes (TI-complexes) based on the monogalactosyldiacylglycerol from different marine macrophytes, were used to enhance the immunogenicity of OmpF-EIII. Also, the chimeric protein incorporated into the most effective TI-complex was used to study its protective activity. The content of anti-OmpF-EIII antibodies was estimated in mice blood serum by enzyme-linked immunosorbent assay (ELISA). To study protective activity, previously immunized mice were infected with TBEV strain Dal'negorsk (GenBank ID: FJ402886). The animal survival was monitored daily for 21 days. OmpF-EIII incorporated into the TI-complexes induced about a 30⁻60- and 5⁻10-fold increase in the production of anti-OmpF-EIII and anti-EIII antibodies, respectively, in comparison with the effect of an individual OmpF-EIII. The most effective vaccine construction provided 60% protection. Despite the dramatic effect on the specific antibody titer, the studied TI-complex did not provide a statistically significant increase in the protection of OmpF-EIII protein. However, our results provide the basis of the future search for approaches to design and optimize the anti-TBEV vaccine based on the OmpF-EIII protein.

Recombinant Fusion Protein Joining E Protein Domain III of Tick-Borne Encephalitis Virus and HSP70 of Yersinia pseudotuberculosis as an Antigen for the TI-Complexes.

Domain III (DIII) of the tick-borne encephalitis virus (TBEV) protein E contains epitopes, which induce antibodies capable of neutralizing the virus. To enhance the immunogenicity of this protein, which has a low molecular weight, the aim of the present work was to express, isolate, and characterize a chimeric protein based on the fusion of the bacterial chaperone HSP70 of Yersinia pseudotuberculosis and EIII (DIII + stem) as a prospective antigen for an adjuvanted delivery system, the tubular immunostimulating complex (TI-complex). The chimeric construction was obtained using pET-40b(+) vector by ligating the respective genes. The resulting plasmid was transformed into DE3 cells for the heterologous expression of the chimeric protein, which was purified by immobilized metal affinity chromatography (IMAC). ELISA, differential scanning calorimetry, intrinsic fluorescence, and computational analysis were applied for the characterization of the immunogenicity and conformation of the chimeric protein. Mice immunization showed that the chimeric protein induced twice the number of anti-EIII antibodies in comparison with EIII alone. In turn, the incorporation of the HSP70/EIII chimeric protein in the TI-complex resulted in a twofold increase in its immunogenicity. The formation of this vaccine construction was accompanied by significant conformational changes in the chimeric protein. Using HSP70 in the content of the chimeric protein represents an efficient means for presenting the main antigenic domain of the TBEV envelope protein to the immune system, whereas the incorporation of this chimeric protein into the TI-complex further contributes to the development of a stronger immune response against the TBEV infection.

Relationship between Adaptive Changing of Lysophosphatidylethanolamine Content in the Bacterial Envelope and Ampicillin Sensitivity of Yersinia pseudotuberculosis.

The low permeability of porin channels is the possible reason for Gram-negative bacterial resistance to antibiotics. The adaptive accumulation of lysophosphatidylethanolamine (LPE) in Yersinia pseudotuberculosis induces conformational changes of OmpF porin that may hinder the transport of antibiotics through this channel. The present study was aimed to test whether the changes in LPE content affect the resistance of bacteria to ampicillin. The addition of glucose to the culture medium was shown to simultaneously increase the level of LPE and minimum inhibitory concentration (MIC) for ampicillin of Y. pseudotuberculosis cells 6- and 2-fold, respectively. However, the coadministration of glucose and polyphenol extract from buckwheat husks reduced the content of LPE 2-fold and restored MIC to the control value. Thus, PBEH can be used as antibiotic adjuvant to improve an antibiotic's ability to cross the outer membrane. The present work demonstrated: (i) the role of adaptive changes in the lipid composition of Y. pseudotuberculosis in the development of antibiotic resistance, and (ii) the promising use of PBEH in combination therapy to increase the susceptibility of Gram-negative bacteria to the conventional ß-lactam antibiotics, probably attenuating in vivo a previously demonstrated effect of LPE on the conformation and function of the OmpF channel.

Modulation of Immunogenicity and Conformation of HA1 Subunit of Influenza A Virus H1/N1 Hemagglutinin in Tubular Immunostimulating Complexes.

The HA1 subunit of the influenza virus hemagglutinin (HA) is a valuable antigen for the development of vaccines against flu due to the availability of most antigenic sites which are conformational. Therefore, a novel adjuvanted antigen delivery system, tubular immunostimulating complexes (TI-complexes) comprising monogalactosyldiacylglycerol (MGDG) from different marine macrophytes as a lipid matrix for an antigen, was applied to enhance the immunogenicity of recombinant HA1 of influenza A H1N1 and to study the relation between its immunogenicity and conformation. The content of anti-HA1 antibodies and cytokines was estimated by ELISA after the immunization of mice with HA1 alone, and HA1 was incorporated in TI-complexes based on different MGDGs isolated from green algae Ulva lactuca, brown algae Sargassum pallidum, and seagrass Zostera marina. Conformational changes of HA1 were estimated by differential scanning calorimetry and intrinsic fluorescence. It was shown that the adjuvant activity of TI-complexes depends on the microviscosity of MGDGs, which differently influence the conformation of HA1. The highest production of anti-HA1 antibodies (compared with the control) was induced by HA1 incorporated in a TI-complex based on MGDG from S. pallidum, which provided the relaxation of the spatial structure and, likely, the proper presentation of the antigen to immunocompetent cells.

Nanoparticulate Tubular Immunostimulating Complexes: Novel Formulation of Effective Adjuvants and Antigen Delivery Systems.

New generation vaccines, based on isolated antigens, are safer than traditional ones, comprising the whole pathogen. However, major part of purified antigens has weak immunogenicity. Therefore, elaboration of new adjuvants, more effective and safe, is an urgent problem of vaccinology. Tubular immunostimulating complexes (TI-complexes) are a new type of nanoparticulate antigen delivery systems with adjuvant activity. TI-complexes consist of cholesterol and compounds isolated from marine hydrobionts: cucumarioside A2-2 (CDA) from Cucumaria japonica and monogalactosyldiacylglycerol (MGDG) from marine algae or seagrass. These components were selected due to immunomodulatory and other biological activities. Glycolipid MGDG from marine macrophytes comprises a high level of polyunsaturated fatty acids (PUFAs), which demonstrate immunomodulatory properties. CDA is a well-characterized individual compound capable of forming stable complex with cholesterol. Such complexes do not possess hemolytic activity. Ultralow doses of cucumariosides stimulate cell as well as humoral immunity. Therefore, TI-complexes comprising biologically active components turned out to be more effective than the strongest adjuvants: immunostimulating complexes (ISCOMs) and complete Freund's adjuvant. In the present review, we discuss results published in series of our articles on elaboration, qualitative and quantitative composition, ultrastructure, and immunostimulating activity of TI-complexes. The review allows immersion in the history of creating TI-complexes.

Production of recombinant porin from Y. pseudotuberculosis in a water-soluble form for pseudotuberculosis diagnostics.

OmpF porin from the outer membrane of Yersinia pseudotuberculosis was cloned into pET-40b(+) plasmid. Using E. coli Rosetta (DE3) strain, MX medium, IPTG concentration of 0.2 mm and post-induction cultivation at 14°C overnight allowed us to obtain a water-soluble form of the recombinant protein (rs-OmpF). Rs-OmpF was shown to have the ordered spatial structure at the levels of secondary and tertiary structure. Rs-OmpF was found to be effective as diagnostic antigen in ELISA for pseudotuberculosis diagnostics.

Engineering of Chimeric Protein Based on E Protein Domain III of Tick- Borne Encephalitis Virus and OmpF Porin of Yersinia pseudotuberculosis.

BACKGROUND: Tick-borne encephalitis poses a serious public health threat in the endemic regions. The disease treatment is restricted to symptomatic therapy, so great expectations are in the development of the prophylactic and therapeutic vaccines. The domain III of E protein of the tickborne encephalitis virus is the main antigenic domain which includes virus-specific epitopes recognized by neutralizing antibodies. OBJECTIVES: The main objective of this study was to design, express, isolate and characterize the chimeric protein based on the fusion of domain III of E protein of the tick-borne encephalitis virus and bacterial porin OmpF from Yersinia pseudotuberculosis. METHODS: The chimeric gene was obtained by the PCR based fusion method from two fragments containing overlapping linker sequences. Resulting plasmids were transformed into BL21(DE3) pLysS electrocompetent cells for subsequent heterologous protein expression. All recombinant proteins were purified using immobilized metal affinity chromatography under denaturing conditions. The identity of the chimeric protein was confirmed by MALDI-TOF mass spectrometry and immunoblot analysis. The content of antibodies against the EIII protein was estimated in mice blood serum by ELISA. RESULTS: The bacterial partner protein was used for decreasing toxicity and increasing immunogenicity of antigen. The chimeric protein was successfully expressed by the Escherichia coli cells. The purified protein was recognized with immunoblots by anti-E protein of tick-borne encephalitis virus monoclonal antibodies. Furthermore, the protein was able to elicit antibody response against domain III of E protein in immunized mice. CONCLUSION: The newly obtained chimeric antigen could be valuable for the development of the preventing tick-borne encephalitis subunit vaccines.